Parkinson's disease (PD) is characterized by the loss of midbrain dopaminergic neurons of the substantia nigra pars compacta (SNpc) and though to a lesser extent dopaminergic neurons of the ventral tegmental area (VTA). SN dopaminergic (DA) neurons project to the striatum and are crucial for locomotor control which is orchestrated by controlled release of dopamine from the projecting DA neurons. Loss of SNpc DA neurons is thought to cause the classical motor symptoms of this disease. However, motivational and affective impairments are also often observed in PD patients. These are usually attributed to a psychological reaction to the general motor impairment and to a loss of some of the neurons within the ventral tegmental area (VTA) (Drui et al., Mol Psychiatry. 2014 March; 19(3):358-67). During fetal development progenitors of these DA neurons are formed in the ventral neural tube of the developing mesencephalon. Progenitor cells from the so-called floor plate region are characterized by expression of the transcription factors FOXA2, Lmx1a and Otx2. These cells give rise to DA SNpc neurons and to DA VTA neurons. Fetal cells dissected form the developing mesencephalon and transplanted to the striatum of Parkinson patients could partially revert the Parkinson symptoms (Barker et al., Lancet Neurol. 2013 January; 12(1):84-91; Kefalopoulou et al., JAMA Neurol. 2014 January; 71(1):83-7). The limited availability of fetal DA progenitors or DA neurons has inspired the development of alternative methods for derivation of functionally equivalent cell populations from alternative sources. Pluripotent stem cells constitute such an alternative cell source. A recent study suggests that DA progenitors differentiated from embryonic stem cells might have the same features as cells derived from the human fetal ventral mesencephalon in terms of i) integration and projection capacity in a rat Parkinson model and ii) functional reversion of Parkinson symptoms (Grealish et al., Cell Stem Cell. 2014 6; 15(5):653-65, Kirkeby et al., Cell Rep. 2012, 1(6):703-14).
Different methods have been reported to support in vitro differentiation of mixed populations of floor plate cells (Kirkeby et al., Cell Rep. 2012, 1(6):703-14; WO2013/067362A1; Doi et al., Stem Cell Reports. 2014 Mar. 6; 2(3):337-50). All protocols based on these publications are performed manually. Protocols comprise complex steps involving harvesting of adherent cells, dissociating cell clumps or embryoid bodies, seeding very defined and specific cell densities, frequent media changes, are conducted in either adherent or suspended culture conditions that may be varied or even alternate within the process.
It is known in the art that in vitro differentiated cell cultures do not constitute a homogenous cell population but may contain a plurality of cells with slightly different characteristics or differentiation stages. However, directed differentiation cultures optimized to generate floor plate cells are considered to contain mainly dopaminergic neuron progenitor cells but may also contain contaminating cells of any kind such as undifferentiated stem cells or a plurality of cells of unwanted differentiation fates such as serotonergic neurons.
Several markers have been disclosed that may serve to isolate subpopulations of dopaminergic progenitors from heterogeneous cell compositions at different time points after initiation of the differentiation process.
WO2013/067362A1 discloses a manually performed method to direct the lineage specific differentiation of human embryonic stem cells (hESC) and/or human induced pluripotent stem cells (hiPSC) into floor plate midbrain progenitor cells and then further into large populations of midbrain fate FOXA2+LMX1A+TH+dopamine (DA) neurons using novel culture conditions as well as CD142 as marker of authentic DA neurons. In contrast, MDAPCs do not express CD142.
Doi et al (Doi et al., Stem Cell Reports. 2014 Mar. 6; 2(3):337-50) described a method for isolation of dopaminergic progenitors using the surface marker corin identifying a subpopulation of cells derived from pluripotent stem cells. Unsorted and sorted corin+ cells were further cultivated and had shown to revert methamphetamine-induced rotation of the rats when transplanted 16 days after corin-based cell sorting. However, corin-based selection only enriches a subpopulation of mesenchephalic floor plate cells (Doi et al., Stem Cell Reports. 2014 Mar. 6; 2(3):337-50), as many FOXA2 positive cells are present in the corin-negative fraction at the day of sort.
WO2013/015457A1 discloses a manually performed method for selecting dopaminergic neuron progenitor cells which comprises detecting a marker specific for dopaminergic neuron progenitor cells. The markers were identified to be expressed in the corin subpopulation, resembling a subpopulation of the mesencephalic floor plate. The markers disclosed therein are markers selected from the group consisting of CD15 (SSEA-1), CD24, CD46, CD47, CD49b, CD57, CD58, CD59, CD81, CD90, CD98, CD147, CD184, Disalogangliosid GD2, SSEA-4, CD49f, SERINC4, CCR9, PHEX, TMPRSS1 IE, HTR1E, SLC25A2, Ctxn3, Cc17, Chrnb4, Chrna3, Kcnv2, Grm2, Syt2, Lim2, Mboat1, St3ga16, Slc39a12, Tacr1, Lrtm1 and Dscam, and/or the negativity of CD201. The preferred marker in this disclosure is Lrtm1 as it can be used together with conventional markers such as corin, Lmx1a, 65B13 and 18A5. Again, all markers disclosed therein are strongly associated to mesencephalic floor plate cells expressing corin, which are a part of mesencephalic floor plate cells only.
Kirkeby et al. (Kirkeby et al., Cell Rep. 2012, 1(6):703-14) were able to generate cell compositions with a high proportion of FOXA2-positive cells from human embryonic stem cells which can currently be regarded to be the most potent candidate cells for therapeutic treatment of Parkinson's disease, since they have been shown to be functionally equivalent to fetal ventral mesencephalic cells by Grealish et al. (Grealish et al., Cell Stem Cell. 2014 6; 15(5):653-65) using the same differentiation protocol as described in Kirkeby et al. (Kirkeby et al., Cell Rep. 2012, 1(6):703-14). Taken together, this analysis shows that hESC-DA neurons were indistinguishable from their fetal counterparts on the basis of graft appearance, morphology, and marker expression 6 months after grafting and that the hESC-derived grafts are rich in both A9-like (SNpc) and A10-like (VTA) DA neurons (Grealish et al., Cell Stem Cell. 2014 6; 15(5):653-65).
Cell compositions with a high proportion of FOXA2+ cells populations were used for these transplantation experiments. However, the degree of differentiation efficiencies reached in these studies rather represent the exception from the rule. It is known in the art that cultivation of a starting cell composition, i.e. pluripotent and/or multipotent stem cells, and the subsequent differentiation process is a highly sophisticated procedure which is subject to user-to-user variation and experimental outcome is influenced by slight variations, such as handling variations in general, density and/or concentration of cells, cytokines, small molecules or other substances used. It is also known in the art, that differentiation efficiency is highly cell-line dependent, i.e. different pluripotent stem cell lines do not exert the same response to the same differentiation protocol. Also experiment-to-experiment variations constitute a big hurdle for standardization of cell production for therapeutic use.
Therefore, there is a need in the art for a method to reproducibly generate a composition of cells, which can be used e.g. in clinical applications such as treatment of Parkinson's disease, wherein the cell composition comprises MDAPCs. This cell composition is the most promising candidate cell population for clinical use in the context of neurodegenerative diseases as it resembles the more complex composition of the ventral mesencephalon of post coitum week 6-7.5 human embryos
Furthermore, generated MDAPCs which are intended to be used in cell therapy have to be produced under GMP or GMP-like conditions. Such a cell-manufacturing process requires extensive training of personnel as well as a dedicated infrastructure, which is a major obstacle to the wider use of these clinical procedures.
Current processes of developing compositions of mesencephalic dopaminergic progenitor cells for cellular therapies have until now been carried out manually.
Therefore, there is a need in the art for a method to reproducibly generate a composition of cells, which can be used e.g. in clinical applications such as treatment of Parkinson's disease, wherein the cell composition comprises MDAPCs.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.